Showing papers on "Growth medium published in 2004"

Uncoupling the effects of abscisic acid on plant growth and water relations. Analysis of sto1/nced3, an abscisic acid-deficient but salt stress-tolerant mutant in Arabidopsis.

Abstract: We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media. sto1 plants were more tolerant in vitro than wild type to Na + and K + both for germination and subsequent growth but were hypersensitive to Li + . Postgermination growth of the sto1 plants on sorbitol was not improved. Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 [ Phaseolus vulgaris ] and to NCED3 GB:AB020817 [Arabidopsis]), a key enzyme in the abscisic acid (ABA) biosynthetic pathway. STO1 transcript abundance was substantially reduced in mutant plants. Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered. Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype. Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation. Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster. The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses.

Defined Anaerobic Growth Medium for Studying Candida albicans Basic Biology and Resistance to Eight Antifungal Drugs

Abstract: The polymorphic fungus Candida albicans is one of the most versatile opportunistic pathogens in humans. Many organs of the human body are potential targets for infection by this pathogen, but infection is commonly localized in the gastrointestinal tract, an environment providing anaerobic growth conditions. We describe a chemically defined anaerobic growth medium for four strains of Candida albicans (A72, SC5314, MEN, and 10261). It is a defined liquid glucose-phosphate-proline growth medium supplemented with oleic acid, nicotinic acid, and ammonium chloride. The cells did not require or respond to added ergosterol. Oleic acid and nicotinic acid are growth factors which are required only for the anaerobic growth of C. albicans. An important technical feature of this study was the use of anaerobically grown inocula to study anaerobic growth. Anaerobically, the cells grew exclusively as mycelia at 25, 30, and 37°C. The doubling time at 30°C was ca. 20 h. The cells did not produce farnesol and did not respond to exogenous farnesol, and they were resistant to the highest tested levels of amphotericin B and four of the azole antifungals. We suggest that the anaerobic growth of C. albicans may contribute to the trailing end point phenomenon and the resistance of C. albicans biofilms to antifungal drugs.

Effect of Medium Components on Bacteriocin Production by Lactobacillus Pentosus ST151BR, a Strain Isolated from Beer Produced by the Fermentation of Maize, Barley and Soy Flour

Abstract: Lactobacillus pentosus ST151BR, isolated from home-brewed beer, produces a 3.0 kDa antibacterial peptide (bacteriocin ST151BR) active against Lactobacillus casei, Lactobacillus sakei, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli. Treatment with Proteinase K or Pronase resulted in loss of activity. Bacteriocin levels of 6400 AU/ml were recorded in MRSbb (De Man-Rogosa-Sharpe broth without Tween 80) at pH 5.5, 6.0 and 6.5. The same growth conditions at pH 4.5 yielded only 1600 AU/ml bacteriocin. Inclusion of Tween 80 in the growth medium reduced bacteriocin production by more than 50%. Growth in the presence of tryptone or tryptone plus meat extract stimulated bacteriocin production, whereas much lower activity was recorded when the bacteria were grown in the presence of meat extract, yeast extract, tryptone plus yeast extract, meat extract plus yeast extract, or a combination of tryptone, meat extract and yeast extract. MRSbb supplemented with maltose, lactose or mannose (2.0%, w/v) yielded bacteriocin levels of 6400 AU/ml. Sucrose or fructose at these concentrations reduced the activity by 50 and 75%, respectively. Growth in the presence of 4.0%(w/v) glucose resulted in 50% activity loss. Glycerol levels as low as 0.1%(w/v) repressed bacteriocin production. Addition of cyanocobalamin, ascorbic acid, thiamine and thioctic acid (1.0 mg/l) to the growth medium did not lead to an increase in bacteriocin production.

LEA (late embryonic abundant)-like protein Hsp 12 (heat-shock protein 12) is present in the cell wall and enhances the barotolerance of the yeast Saccharomyces cerevisiae.

Abstract: Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

Isolation of a β-carotene over-producing soil bacterium, Sphingomonas sp.

Abstract: A carotenoid-accumulating bacterium isolated from soil, identified as a Sphingomonas sp., grew at 0.18 h−1 and produced 1.7 mg carotenoids g−1 dry cell, among which β-carotene (29% of total carotenoids) and nostoxanthin (36%). A mutant strain, obtained by treatment with ethyl methanesulfonate, accumulated up to 3.5 mg carotenoids g−1 dry cell. Accumulation of β-carotene by this strain depended on the oxygenation of the growth medium, with maximal accumulation (89%) occurring under limiting conditions. β-Carotene accumulation could be further enhanced by incubating the cells in the presence of glycerol (either not or only slowly assimilated) and yeast extract resulting in an accumulation of 5.7 mg β-carotene g−1 dry cell wt. The strain used lactose as carbon source with similar biomass and carotenoid production, providing a viable alternative use for cheese whey ultra-filtrate.

Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies.

Abstract: Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 10 8 CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.

Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae

Abstract: Cytochrome P450s are heme-thiolate oxygenases involved in a wide number of reactions such as epoxidation, hydroxylation, and demethylation. Heterologously expressed eukaryotic P450s are potentially useful biocatalysts for stereospecific oxygenation reactions under mild conditions. Numerous factors, such as intracellular pH, cytochrome P450, cytochrome P450 reductase, NADPH, and oxygen concentration all influence the in vivo activity. To systematically examine these factors, we selected ferulate 5-hydroxylase (F5H), a plant P450, with the Saccharomyces cerevisiae WAT11 strain as an expression host. Two media compositions and two cultivation procedures were investigated to optimize the in vivo activity of F5H. We modified a previously published selective growth medium (Pompon et al. [1996] Methods Enzymol 272:51-64) that increased the specific growth rate and cell yield of the host strain. A cultivation procedure with separate growth and induction stages that each contained selective media resulted in a 45% increase of whole cell F5H specific activity. In a medium designed for simultaneous growth and induction, we observed a 2.6-fold higher specific F5H activity, but substantially lower cell yield. Surprisingly, in this medium the higher specific F5H activity did not correlate with a higher P450 concentration. The effects of addition of the first committed heme precursor, delta-aminolevulinic acid, and Fe(III) at the beginning of induction period were also studied for our two-stage procedure. A small, but significant (P < 0.05) increase in whole cell F5H activity was observed following ALA addition.

Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli

Abstract: Aims: To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress. Methods and Results: Cultures of Escherichia coli MG1655 were grown in Luria–Bertani (LB) medium at 37°C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress. A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures. Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy). By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using Δ2–22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx. Conclusions: Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media. Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress. Significance and Impact of the Study: This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology.

Lipid nutrition of Saccharomyces cerevisiae in winemaking

Abstract: Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms. In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen. Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry. Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells. They release lipid biosynthesis metabolites and soon cease to multiply. This paper describes an investigation of the nutritional role of exogenous lipids from inactivated yeast cells (IYCs). Fermentations were carried out in a nitrogen-rich synthetic medium similar to grape juice with glucose and fructose as carbon sources, without lipid sources, and in anaerobiosis. The effect of the addition of IYC was assessed. Cell growth, cell lipid composition, glucose and fructose consumption, and acetic acid production were measured during fermentation. Addition of IYC boosted ...

Production of 3-hydroxydecanoic acid by recombinant Escherichia coli HB101 harboring phaG gene.

Abstract: Heterogenous expression of (R)-3-hydroxydecanoyl-ACP:CoA transacylase gene (phaG) isolated from Pseudomonas putida in Escherichia coli HB101 led to the extracellular production of 3-hydroxydecanoic acid (3HD) in a growth medium consisting of carbon source non-related to 3HD structure, while no 3HD was detected in the growth media inoculated with wild type E. coli HB101 and recombinant E. coli HB101 harboring vector pBluescript SK− only. 3HD production by E. coli HB101 (pLZZGPp) harboring phaG from fructose was 587 mg/l, approximately three times that from cultivation in glucose under same culture conditions. 3HD production was affected by timing of fructose addition and fructose concentration in the culture. As an inhibitor of fatty acid de novo synthesis, the presence of triclosan in the culture could increase 3HD production by E. coli HB101 (pLZZGPp) by about 20–40%. The results further confirmed that (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG) provides 3HD precursors for medium-chain-length polyhydroxyalkanoate synthesis. At the same time, this phenomenon showed that recombinant organisms can be used for production of certain fine chemicals such as hydroxyalkanoic acids.

Carbohydrate carbon sources induce loss of flocculation of an ale-brewing yeast strain.

Abstract: E . V . S O A R E S , A . V R O M A N , J . M O R T I E R , K . R I J S B R A C K A N D M . M O T A . 2004. Aims: To identify the nutrients that can trigger the loss of flocculation under growth conditions in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. Methods and Results: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. Yeast growth with metabolizable carbon sources (glucose, fructose, galactose, maltose or sucrose) at 2% (w/v), induced the loss of flocculation in yeast that had previously been allowed to flocculate. The yeast remained flocculent when transferred to a medium containing the required nutrients for yeast growth and a sole nonmetabolizable carbon source (lactose). Transfer of flocculent yeast into a growth medium with ethanol (4% v/v), as the sole carbon source did not induce the loss of flocculation. Even the addition of glucose (2% w/v) or glucose and antimycin A (0AE 1m g l )1 ) to this culture did not bring about loss of flocculation. Cycloheximide addition (15 mgl )1 ) to glucose-growing cells stopped flocculation loss. Conclusions: Carbohydrates were the nutrients responsible for stimulating the loss of flocculation in flocculent yeast cells transferred to growing conditions. The glucose-induced loss of flocculation required de novo protein synthesis. Ethanol prevented glucose-induced loss of flocculation. This protective effect of ethanol was independent of the respiratory function of the yeast. Significance and Impact of the Study: This work contributes to the elucidation of the role of nutrients in the control of the flocculation cycle in NewFlo phenotype yeast strains.

Comparison of Agar and an Agitated, Thin-film, Liquid System for Micropropagation of Ornamental Elephant Ears

Abstract: Micropropagation of black-stemmed elephant ear (C. esculenta (L.) Schott 'Fon- tanesii')' and upright elephant ear (A. macrorrhizos G. Don) were compared in semi-solid agar media and agitated, liquid thin-fi lm bioreactor vessels at four explant densities (33, 100, 165, and 330 explants/L of media) using two growth regulator combinations: 1) 1 µM benzylaminopurine (BA)—growth medium, and 2) 3 µM BA plus 3 µM ancymidol—multipli- cation medium. The thin-fi lm liquid system outperformed agar culture for most measured responses. Some exceptions were relative dry weights at higher explant densities and multiplication rate of Colocasia. When the thin-fi lm liquid system was compared to agar culture, Alocasia explants produced their greatest biomass and had the least residual sugar at the highest explant density. Alocasia explants multiplied most rapidly and had the greatest relative dry weight on liquid media at the low explant densities. Alocasia plants were larger in growth medium than multiplication medium and larger in liquid medium than agar medium. When compared to agar, Colocasia in the thin-fi lm liquid system produced the greatest biomass at the highest explant density in growth medium, had the greatest relative dry weight at the lowest explant density, and used the most sugar at the highest explant density. Alocasia and Colocasia would likely produce greater fresh and dry weight at the highest explant density if additional sugar were supplied during thin-fi lm culture. Greater growth in thin-fi lm culture of Alocasia and Colocasia is due in part, to greater availability of sugar in liquid compared to agar medium.

Development of Growth Medium for Centella Asiatica Cell Culture Via Response Surface Methodology

Abstract: The effects of sucrose, Indole-3-Acetic Acid (IAA) and 6-BenzylAminoPurine (BAP) concentrations on cell growth of Centella asiatica cell suspension culture were studied. The concentrations were designed using Central-composite experimental design and regression analysis was carried out to obtain response surface model describing cell growth for prediction of optimum conditions. Only sucrose as a single factor was positively significant for cell growth. Increasing sucrose concentration from 3.32 to 6.68% (w/v) resulted in an increase in dry cell weight from 16-27 g L-1. IAA and BAP as single factors and other possible interaction effect were insignificant. The optimum values predicted to be 6.68% (w/v) sucrose, 0.84 mg L-1 IAA and 1.17 mg L-1 BAP yielding 27.4 g L-1 dry cell weight with 81.4% regression equation fitness of the experimental data.

Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium

Abstract: We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4+ and NO3- were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein.

Cheese whey: an alternative growth and protective medium for Rhizobium loti cells.

Abstract: Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at −20 °C and −80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.

Induction of overproducing alkaline protease Bacillus mutants through UV irradiation

Abstract: Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to examine the changes in alkaline protease gene expression following UV irradiation. Induction of mutation in Bacillus mutant strains was carried out by 0, 5, 10, 15 and 20 min. exposure times of UV irradiation and different distances between the treated bacterial cultures and UV source. Results revealed that alkaline protease activity assayed under submerged culture conditions was more accurate than the relative growth production (C/G) method, because there was no proportional correlation between zone diameter and the ability to produce the enzyme in submerged cultures. No enzyme activity was scored with B. pumilus mutants, while the activity was pronounce, in case of B. alvei mutants. Mutants No.15, 55 and 34 were the most efficient in enzyme production under submerged conditions being 68.8, 81.1 and 99 U/ml, respectively. Their alkaline protease activities were 2.6, 3.02 and 3.7 folds than those of the original strains. The optimal enzyme production was achieved after 48 hr at air: medium ratio of 39: 1. Results also proved that the inoculum size in all tested mutant ranges had no significant effect on the enzyme production .The supplementation with glucose to growth medium gave the highest level of enzyme productivity, while lactose showed the lowest. The addition of arabinose and xylose completely inhibited the enzyme production by both tested strains, while incorporation into the culture medium of sucrose and maltose failed to produce the enzyme in B. alvei, but in mutant No.8, they enhanced high levels of productivity.

Method for selectively culturing epithelial or carcinoma cells

Abstract: A method for selectively growing epithelial cells or carcinoma cells in vitro without fibroblast overgrowth comprises (a) suspending a cell pellet comprising digested epithelial or carcinoma cells in a first growth medium, the medium comprising D-valine MEM, methyl cellulose, fetal serum, glutamine and an antibiotic; wherein the methyl cellulose is present in the medium at a concentration sufficient to inhibit growth of fibroblast cells present in the cell pellet; (b) adding the suspension to a cell culture vessel comprising an inner surface which has been at least partially coated with an attachment medium comprising a protein extract, D-valine MEM, glutamine and an antibiotic; and (c) incubating the suspension in the coated vessel to allow selective growth of the epithelial cells or carcinoma cells.

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated, Thin-Film Liquid System for Micropropagation

Abstract: Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.

Barrage Zone Formation Between Vegetatively Incompatible Fusarium graminearum (Gibberella zeae) Isolates.

Abstract: Vegetative compatibility has been used to assess the population biology of many fungal plant pathogens. However, for many species, including Fusarium graminearum, this has meant making auxotrophic mutants to force heterokaryon formation. A method was developed to observe barrage zones of thick, raised mycelium at the junctions of vegetatively incompatible F. graminearum isolates. The appearance of the barrage zones was influenced by the growth medium and the light. Barrage zones on V8 agar were thicker and better defined than those on potato dextrose agar, Spezieller Nahrstoffarmer agar, and water agar. The addition of ground wheat kernels to V8 agar enhanced barrage zone formation. Incubating the cultures under constant light at 2,150 lx produced more distinct barrage zones than constant light at 3,400 lx, constant darkness, or ambient room light. Forty-three F. graminearum isolates from 34 vegetative compatibility groups, determined previously using nit auxotrophic mutants, were paired in all c...

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe.

Abstract: The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

Liquid culture carbon, nitrogen and inorganic phosphate source regulate nematicidal activity by fluorescent pseudomonads in vitro

Abstract: Aims: The aim of the present investigation was to determine the influence of nutrients on the nematicidal activity by Pseudomonas aeruginosa strain IE-6S+ and Ps. fluorescens strain CHA0 in vitro. Methods and Results: Culture filtrate of IE-6S+ and CHA0 obtained from chemically defined medium caused mortality of Meloidogyne javanica juveniles in vitro and that growth medium amended with various C, N or inorganic phosphate (Pi) sources markedly influenced nematicidal activity of the two bacteria. Glycerol (C source), propionate (fatty acid precursor) and l-lysine (N source) enhanced nematicidal activity while glucose (C), l-valine (N) and Pi substantially repressed nematicidal activity of the two bacteria. Conclusion: Liquid culture amendments with various C, N or Pi sources modulate the biosynthesis of nematicidal agents to a different extent in vitro. Significance and Impact of the Study: Developing bacterial strains more responsive to certain environmental signals can be exploited for increased secondary metabolite production in pharmaceutical fermentations and offers new avenues to improve biocontrol.

Release of nucleic acids by eukaryotic cells in tissue culture.

Abstract: Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.

Growth Temperature-Dependent Conversion of De novo-Synthesized Unsaturated Fatty Acids into Polyhydroxyalkanoic Acid and Membrane Cyclopropane Fatty Acids in the Psychrotrophic Bacterium Pseudomonas fluorescens BM07

Abstract: A psychrotrophic bacterial strain, Pseudomonas fluorescens BM07, synthesized unsaturated fatty acids (UFA) from fructose in response to lowering of growth temperature, and incorporated them into both polyhydroxyalkanoic acid (PHA) and membrane lipid. The blocking of PHA synthesis by adding 5 mM 2-bromooctanoic acid to the growth medium, containing 70 mM fructose, was found to be a useful means to profile the composition of membrane lipid by gas chromatography. As the growth temperature changed from 35 to 5°C, the total content of two UFA, 3-hydroxy-cis-5-dodecenoic acid (C 12:1 ) and 3-hydroxy-cis-7-tetradecenoic acid (C 14:1 ), in PHA increased from 31 to 44 mol%. The growth at lower temperatures also led to an increase in the level of two major UFA, palmitoleic acid (C16:1 cis9) and cis-vaceenic acid (C18:1 cis11), in membrane lipid. A fraction of these membrane-lipid UFA was converted to their corresponding cyclopropane fatty acids (CFA). The CFA conversion was a function of culture time, exhibiting biphasic increase before and after entering the stationary phase. However, pH changes in growth media had no effect on the CFA conversion, which is contrary to the case of E. coli reported. The cells grown at 30°C responded to a cold shock (lowering the medium temperature down to 10°C) by increasing the level of C16:1 cis9 and C18:1 cis11 up to that of 10°C-grown control cells and concomitantly decreasing the relative level of cis-9,10-methylenehexadecanoic acid (the CFA converted from C16:1 cis9) from 14 to 8 mol%, whereas the 10-grown cells exhibited little change in the lipid composition when exposed to a warmer environment of 30C for 12 h. Based on this one-way response, we suggest that this psychrotrophic strain responds more efficiently and sensitively to a cold shock than to a hot shock. It is also suggested that BM07 strain is a good producer of two unsaturated 3-hydroxyacids, C 12:1 and C 14:1 .

Cultivation of Marine Sponges: From Sea to Cell

Abstract: Marine sponges are one of the richest natural sources of secondary metabolites with a potential pharmaceutical application. A plethora of chemical compounds, with widely varying carbon skeletons, possessing among other anticancer, antiviral, antibiotic, antiinflammatory and antimalaria activity has been discovered. While for most metabolites their molecular mode of action is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases has been reported. Knowledge on the mode of action is one of the key factors required to transform bioactive compounds into medicines. The rich diversity in bioactive compounds from sponges has provided molecules that interfere with the pathogenesis of a disease at many different points, which increases the chance of developing selective drugs against specific targets (Chapter 2).Unfortunately, these secondary metabolites are usually present in trace amounts, and natural stocks are too small to sustain the development of widely available medicines. The development of ways to obtain large quantities of the secondary metabolites is therefore currently the most important quest. A number of biotechnological methods could potentially provide the required amount of bioactive substances. Three methods were studied in this thesis: Ex situ culture The term ex situ culture refers to cultivation of functional sponges outside of the sea. One of the crucial issues for the ex situ cultivation of sponges is the design of a suitable growth medium. Generally sponges are regarded as particle feeders (bacteria and algae), but they are also capable of the uptake of (partly) dissolved organic carbon sources. The use of powdered substrates can be beneficial for the ex situ culture of sponges under controlled conditions, because an optimal mix of nutrients can be developed and a constant quality can be guaranteed. The ex situ growth rates of sponges cultured on these substrates could be improved, when compared to the sea, but they remain low and resulted in long-term experiments. In order to optimise the growth rate of sponges, it is important to have insight in the way that sponges grow. The suitability of three different models (linear, exponential and radial accretive growth) to describe the growth of both globose and encrusting sponges was assessed. For both morphological appearances, radial accretive growth was the preferred model to simulate the growth. The model can be a valuable tool to make a sound comparison between growth rates of different sponges. In addition, it can be used to study the quantitative effect of factors, such as pressure, light, current, age, temperature or the nutrient source or -concentration on the growth rate of sponges (Chapter 3). Primmorphs Primmorphs are spherical-shaped sponge-cell aggregates with a diameter of approximately 1 mm. They are formed from a dissociated cell suspension under gentle agitation and resemble buds and gemmules, which are the naturally produced asexual regeneration bodies. Primmorph formation seems to be a universal characteristic of marine sponges, as they were obtained from seven different species. By scanning electron microscopy (SEM) it was observed that the primmorphs are very densely packed sphere-shaped aggregates with a continuous pinacoderm (skin cell layer) covered by a smooth, cuticle-like structure. The latter characteristic is probably the reason why primmorphs are more robust than functional sponges and can be easily maintained for a long time. Incubation of primmorphs in a rich medium to attempt cultivation of the aggregates frequently resulted in the growth of bacterial, fungal and eukaryotic unicellular contaminants, which prevented a growth study of primmorphs. The addition of gentamycin or a mixture of penicillin and streptomycin could usually avoid bacterial contaminants, but eukaryotic contaminants were persistent. The addition of the fungicide amphotericin B or a cocktail of antibiotics (kanamycin, gentamycin, tylosin and tetracyclin) prevented the formation of primmorphs (Chapter 4). If primmorphs are actually a kind of experimentally induced regeneration bodies, they could develop into functional sponges. When primmorphs were maintained in seawater enriched with silicate (70 or 150 µM) it was observed that they indeed produced spicules (silica-based skeletal elements) and attached to the bottom of the culture dish, which never occurred at lower silicate concentrations (4 or 25 µM). These results may be explained by available knowledge on the molecular level. Silicate is known to induce the expression of silicatein, the enzyme involved in the production of spicules, at concentrations higher than 60 µM. In addition, silicate has been found to stimulate the biosynthesis of myotrophin, which enhances the production of collagen. Collagen is well known to play an important role in both the attachment of gemmules to a substratum and their subsequent morphogenesis (Chapter 5). Sponge-cell culture Sponge-cell culture may be the tool to overcome the low growth rate, and the corresponding low production rate of the bioactive metabolites of functional sponges. However, the presence of large numbers of associated bacteria, fungi and unicellular organisms inside sponges has been a major obstacle in the development of sponge-cell lines. They have prevented the formation of axenic sponge-cell suspensions, and proliferating sponge cells in cell cultures were therefore looked at with suspicion. For that reason two of prerequisites for the cultivation of sponge cells were developed: A method to distinguish sponge cells in culture from contaminants.A method to assess the viability of cells in culture.The 18S rRNA gene is a suitable marker to identify the origin of eukaryotic cells and a genetic detection method based on this gene was developed for the sponge Dysidea avara . The 18S rRNA gene from a Dysidea avara specimen was sequenced and compared to eukaryotic 18S rDNA sequence(s) that were picked up from a proliferating cell culture that originated from a dissociated Dysidea avara specimen. This method proved to be successful to unambiguously detect whether the cells in culture were actually sponge cells or contaminants (Chapter 6). Cell viability is an essential tool to study the effect of medium components on cell physiology. Especially in case of primary sponge-cell lines it is important to know whether slow growth is caused by a low specific growth rate or by a low viability of the cells. Trypan blue exclusion is a commonly used method to estimate the viability of cell cultures, but for unknown reasons this does not work properly with sponge cells. Therefore, a flow-cytometric viability assay, based on the combined use of fluorescein diacetate (FDA) and propidium iodide (PI) was developed. The effects of temperature, ammonium and the fungicide amphotericin B on the viability of a primary cell culture were studied as examples to assess the suitability of the test. Cell fluorescence measurements based on incubation of cells with FDA or PI, resulted in a good and reproducible estimate of the viability of primary sponge-cell cultures. It was found that the cells rapidly die at a temperature of 22 °C or higher, but that they are insensitive to ammonium concentrations up to 25 mM. Amphotericin B was found to be toxic to the cells (Chapter 7) and this could explain why no primmorphs were formed in the presence of this antibiotic. The current technical status of different methods to produce sponge metabolites was used to study the feasibility of pharmaceuticals from sponges at a large-scale. The production of the metabolites halichondrin B and avarol by chemical synthesis, wild harvest, mariculture, ex situ culture, primmorphs, sponge-cell culture, genetic modification and semi-synthesis were compared on a technical and economical basis, as far as possible. Halichondrin B from a Lissodendoryx sp. and avarol from Dysidea avara were used as model compounds as their products are opposites with respect to their natural concentration inside the sponge. It is concluded that for avarol, which is present in a relatively high concentration, mariculture and ex situ culture could offer feasible methods to compete with currently used medicines against psoriasis. For halichondrin B, the low concentration is a bottleneck for sponge biomass-based production of the compound. A combined approach of (genetically modified) bacterial fermentation (to produce a precursor molecule) followed by a limited number of chemical steps to produce molecules that are derived from sponge chemicals will probably be the most successful method to develop medicines from sponge metabolites that are present in low concentrations (Chapter 8).

Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae.

Abstract: Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1–1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.

Vital culture of sparassic crispa and method of prparing growth medium

Abstract: In the conventional art, it has been a practice to employ complicated procedures using larch sawdust or to use a specific material, a specific strain and, moreover, expensive nutrients. For mass-production by using these methods, an excessively large-scaled equipment is required. Because of using a specific material and a specific strain, it is impossible to produce Sparassic crispa on a mass scale. A tissue is separated from Sparassic crispa occurring in nature and cultured on an agar medium. Then, a seed culture is prepared in a conifer-deciduous sawdust mixture or conifer sawdust. Nutrients are further added to the above-described sawdust medium and the seed culture is transplanted therein. Thus, Sparassic crispa can be grown.

The Presence of Actinidin (Cysteine Protease) and Recombinant Plasmids Carrying the Actinidin Gene Influence the Growth of Saccharomyces Cerevisiae

Abstract: Actinidin is a protease found abundantly in the fruit of Actinidia chinensis or Kiwi fruit. The overproduction of this protein in microorganisms, especially using the yeast Saccharomyces cerevisiae, would be economically valuable as it would simplify the extraction and purification of the protein. It was observed, however, that the yeast growth rate was reduced by the presence of externally supplied actinidin in the growth medium. It was also observed that actinidin present in the yeast growth medium was partially degraded. Several actinidin-encoding gene variants have been cloned in a yeast expression and secretion vector. It was observed that different actinidin gene constructions influenced the growth rate of S. cerevisiae in complete media. Recombinant plasmids carrying only the mature actinidin-encoding DNA sequences reduced yeast transformability significantly and had the least stability. The results thus suggest that the presence of a recombinant plasmid carrying a gene of a potentially toxic protein may result in a deleterious effect on the host cell.